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A visual method for identifying plaques containing λ with recombi-
nant DNA involves the use of the lacZ gene. This gene codes for part of the
β -galactosidase enzyme, an enzyme that cleaves lactose to produce glucose
and galactose. Inserting exogenous DNA into this gene inactivates synthesis
of β -galactosidase. To identify E. coli colonies containing recombinant phage,
the agar is made up with a lactose analog called X-Gal (5-bromo-4-chloro-3-
indolyl- β - d -galactopyranoside). X-Gal is cleaved by β -galactosidase into a prod-
uct that is bright blue. If exogenous DNA has inserted into and disrupted the
β -galactosidase gene, plaques look white or colorless. Plaques containing λ without
the exogenous DNA will produce the blue color.
Figure 6.6 outlines the steps involved in one strategy for producing a repre-
sentative genomic library in a λ replacement vector. The genomic DNA and the
vector DNA can be prepared simultaneously. In this example, the genomic DNA
is cut with a mixture of two restriction endonucleases ( Hae III and Alu I) in such a
manner that the DNA is only partially digested. The genomic DNA is then sized so
that fragments of 20kb are isolated. Meanwhile, the λ vector DNA is digested
with the restriction enzyme Eco RI and purified so that the stuffer fragments are
removed. The genomic DNA has linker molecules added to it before the anneal-
ing reaction. When the genomic DNA and the vector DNA are combined, they
anneal at their complementary cohesive ends and are ligated together. The last
step involves providing a protein coat for the DNA by in vitro packaging.
Commercial cloning kits simplify the procedures considerably because such
kits provide vectors, enzymes, in vitro packaging materials, and detailed proto-
cols. It is even simpler to supply genomic DNA to a company who will provide
a complete genomic library for a fee. However, a few points should be made
about constructing a genomic library.
The genomic DNA to be cloned must be of high molecular weight and not
excessively sheared during its isolation from the insect. High-molecular-weight
DNA is needed because the DNA will be partially digested with a restriction
enzyme to generate a random collection of DNA fragments and the fragments
need to be at least 20 kb. The DNA to be digested actually must be longer than
20 kb so that after digestion both ends of the fragment will have cohesive ends.
DNA fragments with only one cohesive end (and one broken end) cannot be
inserted into vectors. DNA shorter than 20 kb won't be packaged into the phage
and will be lost. Thus, DNA extraction should be carefully executed to avoid
damaging or shearing the DNA.
The genomic DNA fragments ideally will be representative of the entire
insect genome. If the restriction enzymes that are used cut relatively frequently
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